Adapted from Outline of Richard Steinman, M.D., Ph.D

Steinman -1

Hematopoiesis Tony Brickner, Ph.D. 1/10/05 [email protected]

Adapted from outline of Richard Steinman, M.D., Ph.D.

OUTLINE:

Blood as an organ system.

Basic cellular functions important in hematopoiesis and cancer.

Where does blood come from

What are some of the cells in the blood

Stem Cells

Multipotent progenitor Cells

Committed Precursor Cells

Terminally Differentiated Cells

How these work together in hematopoiesis

Cytokines

Transcription factors

How these work together in host defense

What can go wrong in leukemias

Hematopoiesis

Blood as organ system.

One can view the hematopoietic system from a range of metaphorical perspectives, all of which merely hint at its intricacy and dynamism. It is a salad of distinct cells, layered into a richly textured topography within the marrow. It is a frantic dance of erythrocytes squeezing into the bloodstream leaving their nuclei behind, of ribbons of platelets streaming out of lumbering megakaryocytes, of phagocytes tugged forward by their Steinman-2 ruffling membranes in a hunger-crazed can-can. It is a tower of babel in which progenitor cells listen attentively for their siren cytokines within the percolating chemical song. Unyielding and xenophobic, it is intent on the destruction of foreign material or damaged cells; yet it also revels in the emergence of dissimilar lineages from indistinguishable stem cells. And the hematopoietic system is a volcano of production, spewing billions of new neutrophils during the span of this presentation alone. In short, the hematopoietic system is a coordinated network of communication and response, movement, growth and death i.e., it is an organ system.

Basic cellular functions important in hematopoiesis and cancer. The myriad characteristics of the blood described above are choreographed by a balance between four basic cellular events:

Differentiation; Proliferation; Apoptosis; and Movement.

The progression from early, undifferentiated cells to terminally differentiated cells of distinct lineages requires a precise balance between these cellular outcomes. A fraction of a percent change in the level of apoptosis or proliferation relative to differentiation can lead over time to substantial aberration in the number of blood elements, such as neutropenia (low granulocyte count) or leukoproliferative disorders including leukemia.

It has been increasingly recognized that genes which play an important role in suppression of tumors, such as p53, also play a role in maintaining the balance between cell growth, death and differentiation pathways in normal cells.

Where does blood come from? In adult humans, blood is located within the vasculature, and in the axial bone marrow. Cells important in host defense also reside in lymphatic and splenic tissue (lymphocytes), and within tissue (macrophages). Although hematopoiesis takes place in the axial marrow in adults, it is sited in all marrow in children, and in the liver and spleen prenatally or in some diseases (agnogenic myeloid metaplasia). Bone marrow is specially construed to support the proliferation and differentiation of hematopoietic cells. It consists of a honeycombed latticework of venous sinuses. The endothelial cells lining the marrow sinuses are bounded by fibroblast-like stromal cells which generate a extracellular matrix which provides a nurturing microenvironment for hematopoiesis to proceed. The hematopoietic cells are wedged between the vascular sinuses, with megacaryocytes and erythroblasts clustered against the sinuses. Granulocytic differentiation occurs deeper within the hematopoietic space. The close contact between hematopoietic cells and the stroma facilitate transmission of proliferative signals or diffusion of locally-produced cytokines.

Before there was bone marrow, there was the yolk sac. Primitive hematopoiesis takes place in the yolk sac. It is thought that stem cells may migrate from there to the fetal liver.

Blood cells and blood vessels go together like memory and the hippocampus. In fact, some evidence indicates that they are made together--that both derive from a primitive Steinman -3

mesenchymal cell called a hemangioblast. Both blood vessels and the earliest blood cells express a receptor for the VEGF growth factor; without VEGF neither develop. One report indicates that early blood cells produce an endothelial growth factor called angiopoietin to help seduce blood vessels to grow around them.

What are some of the cells in the blood

Mature blood cells can be divided into several categories: lymphoid, monomyelocytic, erythrocytic, and megakaryocytic. In some cases, the terminally differentiated cells in these lineages can still be stimulated to divide-e.g. memory T cells. The terminally differentiated myeloid cells--granulocytes--do not divide. Other final products in the circulation-platelets and erythrocytes are anucleate.

From the standpoint of host defense, the blood system contains two different sort of defenses--innate immunity (eg granulocytes), and adaptive immunity (eg lymphocytes).

It is important to think of what cells do and do not divide, because only dividing cells can give rise to leukemias. One cannot have a leukemia of a neutrophil, for instance, because it is a non-dividing (eg post-mitotic) cell.

A 70 kg individual has roughly 5 liters of blood, of which cells comprise 2.2 liters are cells and serum comprises the rest. The total volume of leukocytes (1.6% of cells) is about 40 ml, and the total volume of circulating platelets is about 7 ml.

The final number of different mature blood types in the blood are highly regulated. A characteristic count could be: cell type cell type % of WBC cells/mm3 range WBC 100% 6000 seg neut 60% 3600 1160-8300 b; 1700-8100 w band neut 2% 120 monocyte 8% 480 200-950 baso 2 120 0-200 eosin 3 180 1-450 RBC 5,000,000 3.7-5.2 E6 plts 300,000 133-333

There are 1000 times as many RBC as WBC in the circulation. However the ratio of RBC to WBC production is only 10-100:1. The higher ratio of RBC:WBC in the circulation results from the longer survival of RBC in the circulation. Steinman-4

Where do they come from?

Blood cell production is highly coordinated to maintain circulating cell numbers within certain levels and to respond rapidly to conditions requiring extra cells. How does this happen?

A lineage diagram has been devised to outline how cells are increasingly restricted in the types of progeny which they can give rise to as differentiation proceeds.

Eo sin

Stem Cells Within our bone marrow, almost as rare as lottery jackpot tickets, are pluripotent stem cells which give rise to all blood lineages (10-5/mononuclear cells). Fewer than 10 such cells, injected into irradiated mice, reconstitute hematopoiesis in their marrow. Steinman -5

The most primitive blood cells are similar to vascular cells. Recent experiments support the view that there is a common precursor for blood vessels and blood cells--a hemangioblast cell.

Stem cells have been found to have several characteristics. They are: pluipotent lack differentiation markers small quiescent

It is hard to find and study stem cells. Two approaches are used to purify them. A surface marker called CD34 is present on most stem cells and is lost in more mature cells. Using a technique called flow cytometry, mononuclear cells can be sorted and cells which express CD34 and do not express CD38 (a marker of more mature cells). This CD34+CD38- population is enriched in cells which exhibit stem cell behavior. Another approach is to select hematopoietic cells which express the receptor for VEGF. This population of cells is very primitive, and may represent some of the first hematopoietic cells to develop from a hemangioblast. These cells are extremely effective in transplantation in mouse models.

Stem cells can also be selected based on their ability to survive in the presence of growth factors and the cytotoxic nucleotide analogue 5FU. In these culture conditions, any cells which begin to proliferate will die. Because early stem cells are deep in dreams of quiescence, they will be enriched in surviving cells.

Two types of functional assays have been used to identify stem cells. One is their ability to give rise to mature lineages for a prolonged period of time in in vitro culture. The other is the ability of the cells stably to generate multiple hematopoietic lineages in irradiated or SCID (immunodeficient) mice. A hematopoietic stem cell is defined most strictly in terms of its ability serially to reconstitute hematopoiesis in such mice. A stem cell loses the ability to do this after it has self-renewed several times.

1° transplant 2° 3° Steinman-6

When cells are transplanted, a CD34+CD38- population of cells supports short term hematopoiesis, following which a CD34-CD38- cell population supports long term reconstitution of the blood system.

If our stem cells have limited capacity, how do we get to live into our vivacious twenties and longer? Current belief is that many of our stem cells stay quiescent for long periods of time. On a stochastic basis, they give rise to cycling stem cells which retain pluripotency and generate offspring.

Recently, there has been an increased appreciation of the plasticity of stem cells. That is, there are cells circulating in the bloodstream which have the capacity to give rise to neurons, muscle (smooth, skeletal, cardiac), intestine, liver, skin, and lung tissue. This new understanding is giving rise to a discipline termed regenerative medicine. While still its infancy, this field is likely to have an enormous scientific and clinical impact.

A Question: What is stem cell cycling? Is this self renewal but not differentiation?

Stem cell cycling refers to stem cells which have decided to divide (anthropomorphically speaking). This is in contrast to even more primitive cells which are quiescent (ie out of the cell cycle). Cycling stem cells can give rise to two progeny at each division. A popular notion has been that one is a "self-renewed" stem cell (ie pluripotent, able to engraft, lacking differentiation markers) and that one is a slightly more differentiated progenitor cell. In fact, it's probably not that simple and sometimes each of the daughter cells may have lost some pluripotentiality and sometimes both may keep it. A stem cell can only "self-renew" a limited number of times before it's primitiveness (ability to give rise to all lineages in a way that can be transplanted) is thought to be lost. Steinman -7

Progenitor cells and precursor cells. Cycling stem cells renew and give rise to more mature multipotent progenitor cells, which are more restricted in the offspring which they will generate. This is associated with tremendous amplification in cell number.

s e l f - r e n e w a l m a c r o p h a g e Mitotic Post-Mitotic erythroid monocytic m o n o c y t e 1 3 p r o m o n o - m o n o c y t e blood 2 C F U - M c y t e b l a s t C F U - C F U - G E M M G M 4 C F U - G pluripotent myeloid p r o m y e l o - m y e l o c y t e m e t a - b a n d s / c y t e m y e l o c y t e g r a n u l o c y t e s stem cell marrow p r o l i f e r a t i o n

d i f f e r e n t i a t i o n 1 - S C F , F L T 3 L , T P O , G C S F , I L 6 , ? L E P T I N 2 - I L - 3 , G M C S F , I L - 6 3 - M - C S F 4 - G - C S F

Progenitor cells

These cells are multipotent They respond best to multiple cytokines This is a compartment which expands the number of cells dramatically. Do not self renew.

These progenitor cells are named by the types of colonies which they give rise to: CFU-LM-Pluripotent cell giving rise to lymphoid and GEMM progenitors. Steinman-8

CFU-GEMM-Multipotent cell giving rise to granulocyte, monocyte, erythroid and megakaryocyte colonies.

CFU-GM--Gives rise to both granulocyte and monocyte colonies.

Progenitor cells grow in specialized areas of the bone marrow, separate from stem cells. Cells interact with the marrow microenvironment; colocalization allows modulation of receptor signals by small amounts of cytokines.

Precursor cells Blast cells committed to unilinear differentiation. Most responsive to one or two cytokines. Still replicate until near terminal differentiation. Do not self renew.

More mature, and more limited precursor cells are: CFU-G, CFU-M, CFU-E and CFU-Baso, giving rise, respectively to granulocytes, monocytes, eosinophils and basophils.

Progeny of these precursors increasingly acquire specific differentiation markers and functions.

Example: Myelopoiesis--formation of granulocytes. Order: Myeloblast, Promyelocyte, Metamyelocyte, Myelocyte, Band, Neutrophil.

A Question: What is the difference between proliferation and repopulation?

Repopulation refers to the ability of stem cells to "repopulate" another marrow-i.e. to successfully transplant a recipient in need of a donor marrow. Proliferation refers just to cell division. For instance, the cytokine IL-3 antagonizes repopulation because it apparently stimulates stem cell progeny (i.e. offspring) to divide--that is, it supports the survival and division of CFU-GEMM and CFU-GM progenitor cells. However, IL- 3 does not support stem cell survival and self-renewal. Therefore, IL-3 moves hematopoiesis forward at the expense of the stem cell compartment (that's why you have other cytokines around, like Stem Cell Factor, which unlike IL-3 support stem cell survival). If IL-3 were predominant, repopulation would be compromised because one would have more cells, but they would be more mature but unable to engraft (that means, to set up new and ongoing hematopoiesis in the marrow--you need stem Steinman -9 cells for that).

Example of differentiation from the Precursor Stage onward: Myeloblast to Granulocyte.

b i l l i o n c e l l s p e r k g 0 . 5 1 0 . 1 4 1 . 9 5

2 . 7 m y e l o b l a s t p r o m y e l o c y t e m y e l o c y t e

m i t o t i c ( 7 . 5 d a y s ) 3 . 6

m e t a m y e l o c y t e

2 . 5 b a n d n e u t r o p h i l

n e u t r o p h i l The stages of neutrophil maturation have been divided based on histologic analysis into: Myeloblast, Promyelocyte, Myelocyte, Metamyelocyte, Band and Mature Neutrophil.

- Increasing development of primary, secondary, tertiary granuoles. Most granule contents serve antibacterial or phagocytic functions

-Increasing phagocytic function. Neutrophil is extremely well adapted to the bacterial killing. They surround microorganisms with pseudopodia which fuse to form a phagosome into which granuole contents are released.

Mitotic and post-mitotic pool

Note that cells develop in two stages. Expansion of cell number occurs as cells in the mitotic, or proliferative pool replicate. Myeloblasts, promyelocytes and myelocytes Steinman-10 undergo mitosis and belong to this pool. It consists of 2.6 x 109 cells/kg. Myelocytes give rise to a large number of differentiated cells which no longer divide, but continue to mature into terminally differentiated cells. This is the post-mitotic (maturation or storage) pool, consisting of metamyelocytes, bands and neutrophils. This pool contains 9 x 109 cells/kg.. It takes roughly 7 days for cells to traverse from pluripotent cells through the proliferative pool and another 5-7 days to complete terminal differentiation.

It has been estimated that there are five cell divisions between the myeloblast and myelocyte stage, and three cell divisions at the myelocyte stage. It takes roughly two weeks to proceed from myeloblasts to mature neutrophils. It takes 5 days from the myelocyte stage until cells enter the blood.

In the setting of infection or stress, maturation time may be shortened, divisions may be skipped, and cells may be released into the bloodstream earlier.

A key concept : The marrow contains a large storage pool of neutrophils which can be reserved for release in a setting of stress, and that the exponential expansion of progenitor cells can be augmented by G-CSF under stress conditions.

Neutrophils and Host Defense

• Most are in the marrow--reserve

• Circulating neutrophils, half in vessel, half adherent to wall

• Diapedese in response to FMLP (N-formyl-methionyl-leucyl-phenylalanine), chemotaxins

Steady flow of neutrophils into superficial tissue, skin, mucosa, lungs needed to prevent infection.

• Bactericidal mechanisms

– Degradative enzymes in granules

– Oxidative killing Steinman -11

• Superoxide generation by NADPH-dependent oxidase – Involves hexose monophosphate shunt, cytochrome b Patients with Chronic Granulomatous Disease lack cytochrome b, cannot generate superoxide, and develop repeated infections.

Cytokines The direction in which differentiation proceeds is influenced, in part by chance, and in part by the cytokines with which cells come in contact. These cytokines support the survival and/or proliferation of certain subsets of cells in the differentiation pool:

Progenitor cells require a minimum of two cytokines to grow effectively; committed precursor cells will grow in response to a single cytokine. In some cases, cytokines have different effects on the same population: for instance Stem Cell Factor will increase the survival of stem cells but will not promote their proliferation; whereas IL-3 will promote proliferation and decrease the repopulating ability of stem cells.

What is the clinical utility of these cytokines? Several of these growth factors have come into clinical usage, such as G-CSF to improve the rate of recovery of patient's neutrophil counts after chemotherapy or marrow transplantation. In some cases it is used to enhance host defense after severe infection. Importantly, G-CSF not only causes expansion of committed myeloid precursor cells, it also increases the sensitivity of cycling Steinman-12 stem cells to other cytokines and increases their entry into the cell cycle. G-CSF for those reasons has been effective in speeding the recovery of normal granulocyte counts in patients who are neutropenic after chemotherapy.

One model of hematopoiesis proposes that specific cytokines induce cells bearing receptors for that cytokine to develop along a specific lineage. There is certainly an additional level of control involved in lineage commitment of cells which bear multiple receptors and which dogpaddle in a pool of available cytokines.

Cytokines signal through specific receptors. MODULAR NATURE OF RECEPTORS--for a number of receptors, proximal cytoplasmic domain is sufficient for proliferation; distal sequences are required for differentiation.

MOST CYTOKINE RECEPTORS LACK INTRINSIC TYROSINE KINASE ACTIVITY. SIGNAL THROUGH CYTOPLASMIC PARTNERS, recruiting shc/ras; shc/PI3K; jak/stat pathway. Steinman -13

SOCS Gene SIE

NUCLEUS

The Stat signaling pathway is important in host defense Steinman-14

m e d i a t e d o p p o s e d b y

R e s p o n s e

S O C S 1 A n t i v i r a l r e s p o n s e ( I F N ) S t a t 1

S O C S 3 , M y e l o p o i e s i s , S t a t 3 , 5

e r y t h r o p o i e s i s

T h 1 r e s p o n s e S t a t 4

I m m u n e s u p p r e s s i o n S t a t 3

( I L 1 0 )

T h 2 r e s p o n s e S t a t 6

Master genes--modulators of normal and leukemic hematopoiesis

Another way of modeling hematopoiesis studies the expression of specific genes which strongly control the direction in which cells develop.

Distinct genes have also been identified which are essential for the transition between hematopoietic stages. Disruption of these master genes in mouse embryos is usually fatal because of disrupted hematopoiesis. Most of these genes code for transcription factors--some, such as pu.1 and C/EBP, appear to control the expression of genes for myeloid growth factor receptors. The figure below sketches some of these genes and the lineages they control. Absence of these genes results in the loss of the lineage under that gene's control. Artificial expression of high levels of one of these master gene products can force a multipotential progenitor to differentiate down a pathway controlled by that gene product. Steinman -15

Leukemia and master genes.

Even though these genes are required for normal hematopoiesis, expression of one of these master genes at the wrong time and place in the hematopoietic tree often results in leukemia. This aberrant expression sometimes results from chromosomal rearrangements which lead to the expression of the master gene product at the wrong time, sometimes as part of a chimeric (fusion) protein with the protein coded for on the other chromosome involved in the rearrangement. Examples include hox genes, AML-1, tal/scl.

Even though leukemia may only occur in one lineage, the derangement usually occurs at the stem cell level. In one example below, the leukemic stem cell first made lymphoid leukemic cells and then myeloid leukemic cells. The master gene which was abnormal was called scl (for stem cell leukemia). This gene was essential for stem cell development and erythrocyte development. Because a chromosomal abnormality, it began to be expressed in T cell progenitors and this resulted in a T cell leukemia.

Many leukemias involve genes critical for early hematopoiesis being expressed in more mature progenitor or precursor cells in which they should normally be silent. In many cases this allows proliferation to continue, while the normal differentiation of those cells is blocked. Steinman-16

megakaryocytes erythrocytes

neutrophils monocytes CFU-GEMM Stem Cell B cell requires lineage SCL T cell lineage =scl S C L t r a n s l o c a t e d i n t o T - c e l l r e c e p t o r l o c u s . I s a b e r r a n t l y expressing t u r n e d o n d u r i n g T - c e l l cell d e v e l o p m e n t . Leukemia

Normal Myeloproliferative Acute Leukemia Hematopoiesis Disorder/CML

Stem Cell

gene x gene x gene x

Progenitors

Precursors

gene x

Differentiated Cells